Final Report Summary - VB (VASCULAR BONE) The European population is ageing and as a result the number of bone-related fractures is increasing as well. After a bone fracture, healing starts immediately and angiogenesis, the formation of new blood vessels from previous ones, is the first and a critical step in this regeneration process. Non or non-delayed union fractures often require the implant of a synthetic bone substitute; however commercially available bone substitutes are mainly osteoconductive, and their pro-angiogenic potential although critical for bone healing is still overlooked. Angiogenesis is a multistep process regulated by chemical and mechanical factors. The most well studied mechanical factor is shear stress induced by the blood flow. Increased blood flow is known to augment blood vessels diameter and stimulate angiogenesis. Extracellular stiffness is a very important mechanical factor as well, involved in many cellular events e.g. stem cell differentiation; however their influence in the angiogenic process remains unclear. The present proposal aims to enlarge our fundamental knowledge on stiffness and angiogenesis in order to rationally develop a pro-angiogenic bone substitute.Angiogenesis encompass cell migration, release of lytic proteins, e.g. matrix metalloproteinases, release of growth factors and tube formation. This project investigated he metabolic activity, gene and protein expression, protein and lipid chemical signature and tube formation of endothelial cells (ECs) exposed to low and high stiffness substrates, with the stiffer substrate matching bone rigidity. The paracrine effect of endothelial cells exposed to different rigidities on human mesenchymal stem cell (hMSC) differentiation into osteoblast-like cells was studied using a co-culture system without direct cell to cell contact. ECs were cultured on polyacrylamide gels coated with collagen I of different stiffnesses fitted on the bottom of a 12-well tissue culture plate and hMSCs were cultured on a polysterene insert also coated with collagen I fitted on top of the well. This project has delivered very well on its anticipated outputs. The most striking results is that stiffness modulates protein activity e.g. β-catenin and as well as growth factors expression. To conclude our understanding regarding the role of extracellular stiffness on angiogenesis was significantly enlarged. This knowledge will contribute for the rationale development of bone specific regenerative approaches, resulting in better and more effective treatments, with being elder people the main target of such benefits.